Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells

References

• Walsh G, Walsh E. Biopharmaceutical benchmarks 2022. Nat Biotechnol. 2022;40(12):1722–14. doi:10.1038/s41587-022-01582-x.
   PubMed Web of Science ®Google Scholar
• Lamanna WC, Holzmann J, Cohen HP, Guo X, Schweigler M, Stangler T, Seidl A, Schiestl M. Maintaining consistent quality and clinical performance of biopharmaceuticals. Expert Opin Biol Ther. 2018;18(4):369–79. doi:10.1080/14712598.2018.1421169.
   PubMed Web of Science ®Google Scholar
• Goetze AM, Schenauer MR, Flynn GC. Assessing monoclonal antibody product quality attribute criticality through clinical studies. MAbs. 2010;2(5):500–07. doi:10.4161/mabs.2.5.12897.
   PubMed Web of Science ®Google Scholar
• Alt N, Zhang TY, Motchnik P, Taticek R, Quarmby V, Schlothauer T, Beck H, Emrich T, Harris RJ. Determination of critical quality attributes for monoclonal antibodies using quality by design principles. Biologicals. 2016;44(5):291–305. doi:10.1016/j.biologicals.2016.06.005.
   PubMed Web of Science ®Google Scholar
• Wei Z, Feng J, Lin HY, Mullapudi S, Bishop E, Tous GI, Casas-Finet J, Hakki F, Strouse R, Schenerman MA. Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus. Anal Chem. 2007;79(7):2797–805. doi:10.1021/ac062311j.
   PubMed Web of Science ®Google Scholar
• Luo Y, Lu Z, Raso SW, Entrican C, Tangarone B. Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcγ receptors. MAbs. 2009;1(5):491–504. doi:10.4161/mabs.1.5.9631.
   PubMed Web of Science ®Google Scholar
• Shaul O. How introns enhance gene expression. Int J Biochem Cell Biol. 2017;91:145–55. doi:10.1016/j.biocel.2017.06.016.
   PubMed Web of Science ®Google Scholar
• Spahr CS, Daris ME, Graham KC, Soriano BD, Stevens JL, Shi SDH. Discovery, characterization, and remediation of a C-terminal Fc-extension in proteins expressed in CHO cells. MAbs. 2018;10(8):1291–300. doi:10.1080/19420862.2018.1511197.
   PubMed Web of Science ®Google Scholar
• Harris C, Xu W, Grassi L, Wang C, Markle A, Hardman C, Stevens R, Miro-Quesada G, Hatton D, Wang J. Identification and characterization of an IgG sequence variant with an 11 kDa heavy chain C-terminal extension using a combination of mass spectrometry and high-throughput sequencing analysis. MAbs. 2019;11(8):1452–63. doi:10.1080/19420862.2019.1667740.
   PubMed Web of Science ®Google Scholar
• Delmar JA, Harris C, Grassi L, Macapagal N, Wang J, Hatton D, Xu W. Monoclonal antibody sequence variants disguised as fragments: identification, characterization, and their removal by purification process optimization. J Pharm Sci. 2022;111(11):3009–16. doi:10.1016/j.xphs.2022.08.002.
   PubMed Web of Science ®Google Scholar
• Choi T, Huang M, Gorman C, Jaenisch1 R. A generic intron increases gene expression in transgenic mice. Mol Cell Biol. 1991;11(6):3070–74. doi:10.1128/MCB.11.6.3070.
   PubMed Web of Science ®Google Scholar
• Shabalina SA, Ogurtsov AY, Spiridonov AN, Novichkov PS, Spiridonov NA, Koonin EV. Distinct patterns of expression and evolution of intronless and intron-containing mammalian genes. Mol Biol Evol. 2010;27(8):1745–49. doi:10.1093/molbev/msq086.
   PubMed Web of Science ®Google Scholar
• Xu DH, Wang XY, Jia YL, Wang TY, Tian ZW, Feng X, Zhang YN. SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells. J Cell Mol Med. 2018;22(4):2231. doi:10.1111/jcmm.13504.
   PubMed Web of Science ®Google Scholar
• Buchman AR, Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988;8(10):4395–405. doi:10.1128/MCB.8.10.4395.
   PubMed Web of Science ®Google Scholar
• Palmiter RD, Sandgren EP, Avarbock MR, Diane Allen D, Brinster RL. Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci U S A. 1991;88(2):478–82. doi:10.1073/pnas.88.2.478.
   PubMed Web of Science ®Google Scholar
• Agarwal N, Ansari A, Copenhaver GP. Enhancement of transcription by a splicing-competent intron is dependent on promoter directionality. PLoS Genet. 2016;12(5):12. doi:10.1371/journal.pgen.1006047.
   Web of Science ®Google Scholar
• Beaulieu E, Green L, Elsby L, Alourfi Z, Morand EF, Ray DW, Donn R. Identification of a novel cell type-specific intronic enhancer of macrophage migration inhibitory factor (MIF) and its regulation by mithramycin. Clin Exp Immunol. 2011;163(2):178–88. doi:10.1111/j.1365-2249.2010.04289.x.
   PubMed Web of Science ®Google Scholar
• Kolasinska-Zwierz P, Down T, Latorre I, Liu T, Liu XS, Ahringer J. Differential chromatin marking of introns and expressed exons by H3K36me3. Nat Genet. 2009;41(3):376–81. doi:10.1038/ng.322.
   PubMed Web of Science ®Google Scholar
• Tilgner H, Nikolaou C, Althammer S, Sammeth M, Beato M, Valcárcel J, Guigó R. Nucleosome positioning as a determinant of exon recognition. Nature Structural & Molecular Biology. 2009;16(9):996–1001. doi:10.1038/nsmb.1658.
   PubMed Web of Science ®Google Scholar
• Millevoi S, Loulergue C, Dettwiler S, Karaa SZ, Keller W, Antoniou M, Vagner S. An interaction between U2AF 65 and CF Im links the splicing and 3′ end processing machineries. Embo J. 2006;25(20):4854–64. doi:10.1038/sj.emboj.7601331.
   PubMed Web of Science ®Google Scholar
• Valencia P, Dias AP, Reed R. Splicing promotes rapid and efficient mRNA export in mammalian cells. Proc Natl Acad Sci U S A. 2008;105(9):3386–91. doi:10.1073/pnas.0800250105.
   PubMed Web of Science ®Google Scholar
• Oganesyan V, Gao C, Shirinian L, Wu H, Dall’acqua WF. Structural characterization of a human Fc fragment engineered for lack of effector functions. Acta Crystallogr D Biol Crystallogr. 2008;64(6):700–04. doi:10.1107/S0907444908007877.
   PubMedGoogle Scholar
• Pertea M, Lin X, Salzberg SL. GeneSplicer: A new computational method for splice site prediction. Nucleic Acids Res. 2001;29(5):1185–90. doi:10.1093/nar/29.5.1185.
   PubMed Web of Science ®Google Scholar
• Persic L, Roberts A, Wilton J, Cattaneo A, Bradbury A, Hoogenboom HR. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene. 1997;187(1):9–18. doi:10.1016/S0378-1119(96)00628-2.
   PubMed Web of Science ®Google Scholar
• Clancy S. RNA splicing | learn science at scitable. Nat Educ. 2008;1(1): 31. https://www.nature.com/scitable/topicpage/rna-splicing-introns-exons-and-spliceosome-12375/.
   Google Scholar
• Bruce SR, Kaetzel CS, Peterson ML. Cryptic intron activation within the large exon of the mouse polymeric immunoglobulin receptor gene: cryptic splice sites correspond to protein domain boundaries. Nucleic Acids Res. 1999;27(17):3446–54. doi:10.1093/nar/27.17.3446.
   PubMed Web of Science ®Google Scholar
• Dominski Z, Kole R. Cooperation of pre-mRNA sequence elements in splice site selection. Mol Cell Biol. 1992;12(5):2108–14. doi:10.1128/MCB.12.5.2108.
   PubMed Web of Science ®Google Scholar
• Liu H, Chumsae C, Gaza-Bulseco G, Hurkmans K, Radziejewski CH. Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC−MS analysis. Anal Chem. 2010;82(12):5219–26. doi:10.1021/ac100575n.
   PubMed Web of Science ®Google Scholar
• Zhang Q, Schenauer MR, McCarter JD, Flynn GC. IgG1 thioether bond formation in vivo. J Biol Chem. 2013;288(23):16371–82. doi:10.1074/jbc.M113.468397.
   PubMed Web of Science ®Google Scholar
• Stoyle CL, Stephens PE, Humphreys DP, Heywood S, Cain K, Bulleid NJ. IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design. Biochem J. 2017;474(18):3179–88. doi:10.1042/BCJ20170342.
   PubMedGoogle Scholar
• Kim SW, Taggart AJ, Heintzelman C, Cygan KJ, Hull CG, Wang J, Shrestha B, Fairbrother WG. Widespread intra-dependencies in the removal of introns from human transcripts. Nucleic Acids Res. 2017;45(16):9503–13. doi:10.1093/nar/gkx661.
   PubMed Web of Science ®Google Scholar
• Murray JI, Voelker RB, Henscheid KL, Warf MB, Berglund JA. Identification of motifs that function in the splicing of non-canonical introns. Genome Biol. 2008;9(6):R97. doi:10.1186/gb-2008-9-6-r97.
   PubMed Web of Science ®Google Scholar
• Berglund JA, Abovich N, Rosbash M. A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev. 1998;12(6):858–67. doi:10.1101/gad.12.6.858.
   PubMed Web of Science ®Google Scholar
• Lefranc M-P, Lefranc G. Immunoglobulins or antibodies: IMGT® Bridging genes, structures and functions. Biomedicines. 2020;8(9):8. doi:10.3390/biomedicines8090319.
   PubMed Web of Science ®Google Scholar
• Watakabe A, Tanaka K, Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993;7(3):407–18. doi:10.1101/gad.7.3.407.
   PubMed Web of Science ®Google Scholar
• Zheng Z-M. Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression. J Biomed Sci. 2004;11(3):278–94. doi:10.1007/BF02254432.
   PubMed Web of Science ®Google Scholar
• Schlatter S, Stansfield SH, Dinnis DM, Racher AJ, Birch JR, James DC. On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog. 2005;21(1):122–33. doi:10.1021/bp049780w.
   PubMed Web of Science ®Google Scholar